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Abstract:

The present disclosure provides methods and compositions for detecting
the presence and activity of creatine transporter proteins (CrT) in
biological samples comprising screening the samples for CrT using
antibodies that bind to the CrT. The methods are useful to detect the
onset of cardiotoxicity in subjects undergoing treatment with
anthrocyclines or those susceptible to heart-related conditions. The
methods and compositions described herein in can be practiced with
diagnostic kits.

Claims:

1. A method for detecting cardiotoxicity in a subject undergoing
treatment with an anthracycline compound comprising: contacting a
biological sample obtained from the subject with a monoclonal antibody
that is specific for the creatine transporter protein; and determining
the level of binding of the antibody to the creatine transporter protein
in the biological sample; wherein a lowered binding of antibody as
compared to a control is indicative of cardiotoxicity.

2. The method of claim 1, wherein the antibody recognizes the isoform of
creatine transporter protein that is recognized by the antibody produced
by hybridoma cell line 4B9 or 8A6.

3. The method of claim 1, wherein the antibody is specific for an epitope
in the first 60 amino acids of the human creatine transporter protein
(SEQ ID NO: 1).

4. The method of claim 1, wherein the antibody comprises antibody
produced by hybridoma cell line 4B9 or hybridoma cell line 8A6.

5. The method of claim 1, wherein the level of antibody binding is
determined by one or more of flow cytometry, immunohistochemistry, ELISA,
or Western blotting.

6. The method of claim 1, wherein the control is a control sample
obtained from a biological sample from an individual not undergoing
treatment with an anthracycline compound, the method comprising
contacting the control sample with the monoclonal antibody that is
specific for the creatine transporter protein; and determining the level
of binding of the antibody to the creatine transporter protein in the
control sample.

7. The method of claim 1, wherein the control is the biological sample
obtained from the individual prior to beginning treatment with the
anthracycline compound.

9. A method for detecting cardiotoxicity in a subject susceptible to a
heart-related condition comprising: contacting a biological sample
obtained from the subject with a monoclonal antibody that is specific for
the creatine transporter protein; and determining the level of binding of
the antibody to the creatine transporter protein in the biological
sample; wherein a lowered binding of antibody as compared to a control is
indicative of cardiotoxicity.

10. The method of claim 9, wherein the antibody recognizes the isoform of
creatine transporter protein that is recognized by the antibody produced
by hybridoma cell line 4B9 or 8A6.

11. The method of claim 9, wherein the antibody is specific for an
epitope in the first 60 amino acids of the human creatine transporter
protein (SEQ ID NO: 1).

12. The method of claim 9, wherein the antibody comprises antibody
produced by hybridoma cell line 4B9 or hybridoma cell line 8A6.

13. The method of claim 9, wherein the level of antibody binding is
determined by one or more of flow cytometry, immunohistochemistry, ELISA,
or Western blotting.

14. The method of claim 9, wherein the control is a sample obtained from
a biological sample from an individual not susceptible to a heart-related
condition, the method comprising contacting the control sample with the
monoclonal antibody that is specific for the creatine transporter
protein; and determining the level of binding of the antibody to the
creatine transporter protein in the control sample.

15. The method according to claim 9 wherein the biological sample
comprises blood or body tissue.

16. A kit for determining the likelihood of the presence of
cardiotoxicity in a subject, the kit comprising (i) an antibody that
specifically binds to the creatine transporter protein and (ii)
instructions for use in a method of determining the likelihood of the
presence of cardiotoxicity in a subject, the method comprising:
contacting a biological sample obtained from the subject with a
monoclonal antibody that is specific for the creatine transporter
protein; and determining the level of binding of the antibody to the
creatine transporter protein in the biological sample, wherein a lowered
binding of antibody as compared to a control is indicative of
cardiotoxicity.

17. The kit of claim 16, wherein the antibody recognizes the isoform of
creatine transporter protein that is recognized by the antibody produced
by hybridoma cell line 4B9 or 8A6.

18. The kit of claim 16, wherein the antibody is specific for an epitope
in the first 60 amino acids of the human creatine transporter protein
(SEQ ID NO: 1).

19. The kit of claim 16, wherein the antibody comprises antibody produced
by hybridoma cell line 4B9 or hybridoma cell line 8A6.

20. The kit of claim 16, comprising one or more of a buffer, a secondary
antibody, or a detection reagent.

21. A method for detecting the onset of cardiotoxicity in a subject
undergoing treatment with an anthracycline compound comprising: obtaining
a blood sample from a subject prior to treatment of the subject with an
anthracycline compound and measuring creatine transporter protein
activity in the blood sample erthyrocytes; obtaining a blood sample from
the subject after receiving at least one treatment with the anthracycline
compound and measuring creatine transporter protein activity in the blood
sample erthyrocytes; comparing the level of creatine transporter protein
activity in the erthyrocytes, wherein a reduced creatine transporter
protein activity in the erthyrocytes after treatment with the
anthracycline compound is indicative of the onset of cardiotoxicity; and
ceasing treatment with the anthracycline compound when the reduced
creatine transporter protein activity is observed.

Description:

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application
No. 61/494,654, filed Jun. 8, 2011, the disclosure of which is hereby
incorporated by reference in its entirety.

FIELD

[0002] The presently disclosed subject matter relates to compositions and
methods for detecting cardiotoxicity, and more specifically, for
determining a reduction in the presence and/or function of creatine
transporter protein.

BACKGROUND

[0003] Anthracyclines are a family of drugs that are effective
anti-neoplastic agents, and are commonly used to treat cancer, including
leukemia, soft tissue sarcomas, and breast and lung cancer.
Anthracyclines intercalate into DNA and are described as topoisomerase
Type II poisons. The anthracycline family comprises, daunomycin,
daunorubicin, doxorubicin/adriamycin, epirubicin, and idarubicin. While
the anthracyclines are known to be potent anti-tumor drugs, their use has
been limited due to potentially life-threatening cardiotoxicity
associated therewith. This problem may be described as cumulative
dose-dependent cardiotoxicity, which can ultimately result in congestive
heart failure.

[0006] The myocardium's main source of Cr is provided by transmembrane
uptake via a specific Cr transporter (CrT), which belongs to the SLC6
family of membrane proteins (Nash S. et al., Receptors Channels 2:
165-174, 1994). One cell line that is useful for studying the effects of
DOX on CrT function is rat neonatal cardiomyocytes (RNCM), as this line
is a primary cardiac cell culture, and thus possesses native cellular
signaling cascades. RNCM preferentially use glucose and lactate as energy
sources over fatty acids, the main energy substrate of adult
cardiomyocytes (Lopaschuk G. D. & Jaswal J. S., J Cardiovasc Pharmacol
56: 130-140, 2010). In addition, HL-1 cells are a well-established
immortalized murine atrial cell line that retain the essential hallmarks
of primary cardiomyocytes, and are a proven expression system to study
protein structure, function and modulation of cardiomyocytes (White S. M.
et al., Am J Physiol Heart Circ Physiol 286: H823-829, 2004). HL-1 cells
have very low native Cr transport and, thus, are ideally suited to study
Cr transport following transfection with a mammalian expression vector
encoding the human CrT.

[0007] Accordingly, it is an object of the present disclosure to provide
methods for detecting the onset of cardiotoxicity in subjects undergoing
treatment with anthrocyclines or those susceptible to heart-related
conditions.

SUMMARY

[0008] One aspect of the present disclosure provides methods for detecting
cardiotoxicity in a subject undergoing treatment with an anthracycline
compound comprising contacting a biological sample obtained from the
subject with a monoclonal antibody that is specific for the creatine
transporter protein; and determining the level of binding of the antibody
to the creatine transporter protein in the biological sample, wherein a
lowered binding of antibody as compared to a control is indicative of
cardiotoxicity.

[0009] Another aspect of the present disclosure provides a method for
detecting cardiotoxicity in a subject susceptible to a heart-related
condition comprising contacting a biological sample obtained from the
subject with a monoclonal antibody that is specific for the creatine
transporter protein; and determining the level of binding of the antibody
to the creatine transporter protein in the biological sample, wherein a
lowered binding of antibody as compared to a control is indicative of
cardiotoxicity.

[0010] Another aspect of the present disclosure provides a kit for
determining the likelihood of the presence of cardiotoxicity in a
subject, the kit comprising (i) an antibody that specifically binds to
the creatine transporter protein and (ii) instructions for use in a
method of determining the likelihood of the presence of cardiotoxicity in
a subject, the method comprising contacting a biological sample obtained
from the subject with a monoclonal antibody that is specific for the
creatine transporter protein and determining the level of binding of the
antibody to the creatine transporter protein in the biological sample,
wherein a lowered binding of antibody as compared to a control is
indicative of cardiotoxicity.

[0011] In certain embodiments, the antibody comprises antibody produced by
hybridoma cell line 4B9. In other embodiments, the antibody comprises
antibody produced by hybridoma cell line 8A6. In other embodiments, the
antibody recognizes the isoform of creatine transporter protein that is
recognized by the antibody produced by hybridoma cell line 4B9 or 8A6. In
other embodiments, the antibody is specific for an epitope in the first
60 amino acids of the human creatine transporter protein (SEQ ID NO: 1).

[0012] In other embodiments, the level of antibody binding is determined
by flow cytometry, immunohistochemistry, ELISA or Western blotting.

[0013] In yet another embodiment, the control is a control sample obtained
from a biological sample from an individual not undergoing treatment with
an anthracycline compound, the method comprising contacting the control
sample with the monoclonal antibody that is specific for the creatine
transporter protein; and determining the level of binding of the antibody
to the creatine transporter protein in the control sample.

[0014] In other embodiments, the biological sample comprises blood or body
tissue.

[0015] Another aspect of the present disclosure provides a method for
detecting the onset of cardiotoxicity in a subject undergoing treatment
with an anthracycline compound comprising obtaining a blood sample from a
subject prior to treatment of the subject with an anthracycline compound
and measuring creatine transporter protein activity in the blood sample
erthyrocytes; obtaining a blood sample from the subject after receiving
at least one treatment with the anthracycline compound and measuring
creatine transporter protein activity in the blood sample erthyrocytes;
comparing the level of creatine transporter protein activity in the
erthyrocytes, wherein a reduced creatine transporter protein activity in
the erthyrocytes after treatment with the anthracycline compound is
indicative of the onset of cardiotoxicity; and ceasing treatment with the
anthracycline compound when the reduced creatine transporter protein
activity is observed.

[0016] Other aspects and advantages will become apparent to those skilled
in the art from a review of the following description that proceeds with
reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are not intended to be limiting in any
way. The foregoing objects, features and advantages of the present
disclosure will become more apparent from a reading of the following
description in connection with the accompanying drawings.

[0022] FIG. 4 shows immunoblots of CrT protein expressed in HL-1 cells.
Representative Western blots of solubilized protein isolated from HL-1
cells expressing a negative control, CrT, CrT-myc or a rat-human CrT
chimera are shown. The immunoblot was probed with one of 2 distinct
anti-human CrT rat monoclonal antibody clones (4B9 or 8A6), or myc tag
antibody, as described herein in Examples 1 and 2. Equal amounts (25
μg) of protein were loaded in each lane. TATA binding protein (TBP)
was used as a loading control.

[0023] FIG. 5A and FIG. 5B show that DOX reduces cell-surface CrT protein
content in HL-1 cells. FIG. 5A shows the abundance of CrT cell-surface
protein that was analyzed using cell biotin labeling, followed by
isolation using avidin affinity and Western blots probed with 4B9
antibody. FIG. 5B shows the intensity of the bands quantified using IMAGE
J. Data represent the mean±SEM of 4 independent experiments, and an
"*" indicates a significant difference compared with the controls
(p<0.05, ANOVA, Fisher's LSD).

[0030] For the purposes of promoting an understanding of the principles of
the present disclosure, reference will now be made to preferred
embodiments and specific language will be used to describe the same. It
will nevertheless be understood that no limitation of the scope of the
disclosure is thereby intended, such alteration and further modifications
of the disclosure as illustrated herein, being contemplated as would
normally occur to one skilled in the art to which the disclosure relates.

[0031] Articles "a" and "an" are used herein to refer to one or to more
than one (i.e. at least one) of the grammatical object of the article. By
way of example, "an element" means at least one element and can include
more than one element.

[0032] Unless otherwise defined, all technical terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art
to which this disclosure belongs.

[0033] In general, the methods described herein include evaluating the
levels of human creatine transporter protein (CrT) in a biological sample
from a subject. These levels can provide diagnostic and/or prognostic
information, e.g., indicating whether a subject has onset of
cardiotoxicity, either caused by anthracycline treatment or other
heart-related conditions.

[0034] In one embodiment of the presently disclosed subject matter, a
method is provided for detecting cardiotoxicity in a subject undergoing
treatment with an anthracycline compound comprising: contacting a
biological sample obtained from the subject with a monoclonal antibody
that is specific for the creatine transporter protein; and determining
the level of binding of the antibody to the creatine transporter protein
in the biological sample; wherein a lowered binding of antibody as
compared to a control is indicative of cardiotoxicity.

[0035] In one embodiment, the control is a control sample obtained from a
biological sample from an individual not undergoing treatment with an
anthracycline compound, the method comprising contacting the control
sample with the monoclonal antibody that is specific for the creatine
transporter protein; and determining the level of binding of the antibody
to the creatine transporter protein in the control sample. In one
embodiment, the control is the biological sample obtained from the
individual prior to beginning treatment with the anthracycline compound.

[0036] In one embodiment of the presently disclosed subject matter, a
method is provided for detecting cardiotoxicity in a subject susceptible
to a heart-related condition comprising: contacting a biological sample
obtained from the subject with a monoclonal antibody that is specific for
the creatine transporter protein, and determining the level of binding of
the antibody to the creatine transporter protein in the biological
sample, wherein a lowered binding of antibody as compared to a control is
indicative of cardiotoxicity.

[0037] In one embodiment the control is a sample obtained from a
biological sample from an individual not susceptible to a heart-related
condition, the method comprising contacting the control sample with the
monoclonal antibody that is specific for the creatine transporter
protein, and determining the level of binding of the antibody to the
creatine transporter protein in the control sample.

[0038] Hence, changes in creatine metabolism manifested through
alterations in creatine transport capacity, i.e., changes in creatine
transporter protein abundance and/or function, can be a useful diagnostic
or predictive tool in monitoring the onset of cardiotoxicity in a
subject.

[0039] As used herein, the term "anthracycline compound" refers to the
class of drugs used in cancer chemotherapy derived from Streptomyces
bacteria. Examples include, but are not limited to, daunorubicin,
doxorubicin (Adriamycin), Epirubicin, Idarubicin, Valrubicin and
Mitoxantrone. The term "anthracycline treatment" refers to the use of
these compounds in a cancer regimen.

[0040] As used herein, the term "cardiotoxicity" refers to any condition
where there is damage to the heart muscle. Such damage may be caused by
chemotherapy drugs (e.g., anthracycline compounds) or other medications,
as well as cardiac muscle damage caused by other conditions, such as
hypertrophy, ischemia, ischemia-reperfusion, hypoxia and the like
commonly associated with cardiac arrest, heart failure, etc.

[0041] As used herein, the term "heart-related conditions" refers to those
conditions associated with, or prognosticator of, damage to the heart
muscle. Such conditions include, but are not limited to, high blood
pressure, high cholesterol, smoking, diabetes, age, race and the like.

[0042] As used herein, the term "subject" and "patient" are used
interchangeably herein and refer to both human and nonhuman animals. The
term "nonhuman animals" of the disclosure includes all vertebrates, e.g.,
mammals and non-mammals, such as nonhuman primates, sheep, dog, cat,
horse, cow, chickens, amphibians, reptiles, and the like. Preferably, the
subject is a human patient. More preferably, the subject is a human
patient undergoing treatment with an anthracycline compound or is
susceptible to a heart-related condition.

[0043] Thus, for a subject who has low levels of CrT as compared to
controls, there is a higher probability of cardiotoxicity. Conversely,
for a subject who has normal levels of CrT as compared to controls, there
is a low probability of cardiotoxicity. For example, a subject undergoing
a cancer treatment regimen with an anthracycline compound that exhibits a
low CrT level suggests that anthracycline-induced cardiotoxicity is
highly probable. This diagnosis therefore allows a caregiver to alter
and/or stop treatment before a cardiotoxic event takes place. Such action
allows for the caregiver to design and implement a "personalized"
anthracyclin compound treatment regimen. Similarly, a subject that is
susceptible to a heart-related condition that exhibits a low CrT level
would indicate that a cardiotoxic event is imminent, thereby allowing the
caregiver to take appropriate action.

[0044] Evaluating the levels of CrT in a subject typically includes
obtaining a biological sample. As used herein, the term "biological
sample" refers to any sample that can be obtained from the subject that
comprises CrT protein. Such samples include, but are not limited to,
serum, blood, plasma, urine, or body tissue (e.g., a biopsied tissue
specimen). Levels of the CrT protein in the sample can be determined by
measuring levels of the CrT protein in the sample using methods known in
the art and/or described herein, e.g., immunoassays such as enzyme-linked
immunosorbent assays (ELISA), immunohistochemistry, Western blotting and
the like. In one embodiment, the sample is a blood sample, and the levels
of CrT protein are determined for the erythrocytes from the blood sample.

[0045] For example, a method described herein, e.g., for levels of CrT in
a subject undergoing treatment with an anthracycline compound, can
include contacting a sample from the subject, e.g., a sample including
blood, serum, plasma, urine, or body tissue, from the subject with an
antibody that specifically binds to the CrT protein as described herein.
The methods can also include contacting a sample from a control subject,
normal subject, or normal tissue or fluid from a normal subject with the
antibody to provide a reference or control. Moreover, the method can
additionally include comparing the specific binding of the antibody to
the test subject with the specific binding of the antibody to the normal
subject, control subject, or normal tissue or fluid from the normal or
control subject. Expression from a control subject or control sample can
be provided as a predetermined value, e.g., acquired from a statistically
appropriate group of control subjects.

[0046] An antibody that "binds specifically to" the CrT protein, binds
preferentially to the CrT protein in a sample containing other proteins.
The term "antibody" as used herein refers to an immunoglobulin molecule
or immunologically active portion thereof, i.e., an antigen-binding
portion. Examples of immunologically active portions of immunoglobulin
molecules include F(ab) and F(ab')2 fragments which can be generated
by treating the antibody with an enzyme, such as pepsin. The antibody can
be polyclonal, monoclonal, recombinant, e.g., a chimeric or humanized,
fully human, non-human, e.g., murine, monospecific, or single chain
antibody. In one embodiment, it has effector function and can fix
complement. In one embodiment, the antibody is a rat monoclonal antibody
specific for the CrT protein. In one embodiment, the antibody is 4B9 or
8A6. In one embodiment, the antibody recognizes the isoform of creatine
transporter protein that is recognized by the antibody produced by
hybridoma cell line 4B9 or 8A6. In one embodiment, the antibody is
generated by using the first 60 amino acids of the human creatine
transporter protein (SEQ ID NO: 1) as the antigen. In one embodiment, the
antibody is specific for an epitope in the first 60 amino acids of the
human creatine transporter protein (SEQ ID NO: 1). In one embodiment, the
antibody comprises antibody produced by hybridoma cell line 4B9 or
hybridoma cell line 8A6.

[0047] Detection can be facilitated by coupling (i.e., physically linking)
the antibody or probe to a detectable substance (i.e., antibody
labeling). Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of suitable
enzymes include, but are not limited to, horseradish peroxidase, alkaline
phosphatase, β-galactosidase, or acetylcholinesterase; examples of
suitable prosthetic groups include, but are not limited to, complexes
such as streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include, but are not limited to, umbelliferone,
fluorescein, fluorescein, isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride, quantum dots, or
phycoerythrin; an example of a luminescent material includes, but is not
limited to, luminol; examples of bioluminescent materials include, but
are not limited to, luciferase, luciferin and acquorin, and examples of
suitable radioactive materials include, but are not limited to,
125I, 131I, 35S or 3H.

[0049] In one embodiment, the presently disclosed subject matter provides
a kit for determining the likelihood of the presence of cardiotoxicity in
a subject, the kit comprising (i) an antibody that specifically binds to
the creatine transporter protein and (ii) instructions for use in a
method of determining the likelihood of the presence of cardiotoxicity in
a subject, the method comprising: contacting a biological sample obtained
from the subject with a monoclonal antibody that is specific for the
creatine transporter protein; and determining the level of binding of the
antibody to the creatine transporter protein in the biological sample,
wherein a lowered binding of antibody as compared to a control is
indicative of cardiotoxicity.

[0050] The diagnostic kits of the present disclosure can be configured for
professional (e.g., clinical) or personal use. In one embodiment, the kit
includes an antibody for the detection of CrT protein, as well as a
reagent comprising a binding composition for the detection of the CrT
protein, and instructions for use in a method described herein. A control
can also be included, e.g., an epitope of CrT. In one embodiment, the kit
comprises one or more of a buffer, a secondary antibody, or a detection
reagent.

[0051] Kits generally comprise the following major elements: packaging,
reagents comprising antibodies described above, optionally a control, and
instructions. Packaging may be a box-like structure for holding a vial
(or a number of vials) containing the antibodies, a vial (or number of
vials) containing a control, and instructions for use in a method
described herein. Individuals skilled in the art can readily modify the
packaging to suit individual needs. The foregoing kits can also be
provided with tools to assist in the collection of biological samples.
Some samples of tools include sample containers, such as vials or cups;
needles and syringes for blood or serum samples; antiseptic prepatory
pads; gauze pads; and/or swabs for tissue samples.

[0052] As one example, the kit can contain an antibody that binds
specifically to CrT (e.g., 4B9 and/or 8A6), or an antibody or antigen
binding fragment thereof that binds specifically to the CrT protein. In
one embodiment, the antibody recognizes the isoform of creatine
transporter protein that is recognized by the antibody produced by
hybridoma cell line 4B9 or 8A6. In one embodiment, the antibody is
generated by using the first 60 amino acids of the human creatine
transporter protein (SEQ ID NO: 1) as the antigen. In one embodiment, the
antibody is specific for an epitope in the first 60 amino acids of the
human creatine transporter protein (SEQ ID NO: 1). In one embodiment, the
antibody comprises antibody produced by hybridoma cell line 4B9 or
hybridoma cell line 8A6, or an antibody or antigen binding fragment
thereof that binds specifically to the CrT protein.

[0053] In other embodiments, other methods of detection can be used, e.g.,
colorimetric assays, radioimmunoassays, or chemiluminescent assays.
Sandwich assays can be used as well, e.g., using two monoclonal
antibodies, one labeled with 125I and the other adsorbed onto beads
(see, e.g., the IRMA-BNP2 kit from CISBIO International (France) and
SHIONORIA BNP or ANP kits (SHIONOGI USA, Inc.).

[0054] In one embodiment, the presently disclosed subject matter provides
a method for detecting the onset of cardiotoxicity in a subject
undergoing treatment with an anthracycline compound comprising: obtaining
a blood sample from a subject prior to treatment of the subject with an
anthracycline compound and measuring creatine transporter protein
activity in the blood sample erthyrocytes; obtaining a blood sample from
the subject after receiving at least one treatment with the anthracycline
compound and measuring creatine transporter protein activity in the blood
sample erthyrocytes; comparing the level of creatine transporter protein
activity in the erthyrocytes, wherein a reduced creatine transporter
protein activity in the erthyrocytes after treatment with the
anthracycline compound is indicative of the onset of cardiotoxicity; and
ceasing treatment with the anthracycline compound when the reduced
creatine transporter protein activity is observed.

[0055] The disclosure may be better understood by reference to the
following non-limiting Examples, which are provided as exemplary of the
disclosure. The following examples are presented in order to more fully
illustrate the preferred embodiments of the disclosure and should in no
way be construed, however, as limiting the broad scope of the disclosure.

[0059] Human CrT-cDNA was obtained from Dr. Marc Caron (Duke University,
Department of Cell Biology). An EcoR I site followed by a Kozak sequence
was added to the 5' end, and an Hind III site was introduced at the 3'
end and subcloned into the pcDNA3.1(-) mammalian expression vector
(INVITROGEN, Carlsbad, Calif.). In a separate construct, an XbaI site was
introduced at the 3' end, removing the natural stop codon. This modified
cDNA was then subcloned in the mammalian expression pcDNA 3.1B vector
(INVITROGEN, Carlsbad, Calif.). Transfection of this plasmid resulted in
expression of CrT protein bearing a C-terminal Myc/His tag that did not
alter CrT function and was used to detect the CrT-myc protein by Western
blotting. As a negative control for expression, the cDNA encoding CrT was
subcloned into pcDNA3.1(+) in reverse orientation with respect to the
cytomegalovirus (CMV) promoter.

[0060] The rat/human chimeric construct was prepared in two steps using
polymerase chain reactions. The first step consisted of two reactions. In
the first reaction, the rat N-terminal sequence was amplified using
primers that resulted in a 200-bp fragment, including an EcoR I site, a
Kozak sequence, and the 5' end of the cDNA for rat CrT (forward primer:
5'-gaattccaccatggccaagaagagcgcc-3'), with the 3' end corresponding to the
human sequence at position bp167 of the human cDNA (reverse primer:
5'-catgatgaagtccatctgcctggtcca-3') end. The template for these reactions
was a synthetic DNA fragment, encoding the rat CrT N-terminus. A second
reaction, using the human CrT-cDNA as template, generated a 700-bp DNA
fragment that was complementary at the 5' end (forward primer:
5'-tggacgcgccagatggacttcatcatg-3') to the 3' end of the 200-bp fragment
described above, and encompassed a unique Pst I site at position 1104 of
the human cDNA (reverse primer: 5'-ccctgactctgccaccttgga-3').

[0061] The products of these two reactions were used as templates in the
second step; the forward primer corresponded to the 5' end of the rat CrT
N-terminus, and the reverse primer was the same used in the second
reaction of the first amplification round. The resulting 1120-bp fragment
was subcloned into the pSCB vector (INVITROGEN, Carlsbad Calif.) and
sequenced to verify the integrity of the construct. The EcoR Ito Pst I
fragment was isolated and swapped with the corresponding fragment from
the human full-length open reading frame. The integrity of the construct
was again verified by overlapping sequencing.

[0064] Transient transfection of HL-1 cardiomyocytes was performed using
LIPOFECTAMINE 2000 reagent (INVITROGEN, Carlsbad Calif.) according to the
manufacturer's instructions. Cultures intended for Cr transport
measurements were grown in 24-well culture plates, whereas those used to
characterize the CrT protein were cultured in 6-well dishes. Cells were
allowed to grow for approximately 16 hrs after transfection before they
were incubated with DOX.

[0065] DOX Incubation and 14C--Cr Transport in RNCM and HL-1 Cells:

[0066] 100 nmol/L of DOX represents the average steady-state concentration
of the drug that is typically measured in patient plasma. For
dose-response experiments, cultures were incubated in media containing 25
nmol/L, 50 nmol/L, 75 nmol/L, and 100 nmol/L DOX (SIGMA, St. Louis, Mo.)
for 48 hrs. Time course experiments were conducted with cells incubated
for 4 hrs, 12 hrs, 24 hrs, 36 hrs, and 48 hrs in media containing 100
nmol/L DOX. To determine the recovery of Cr transport following DOX
exposure, cells were first incubated in the presence of 100 nmol/L DOX
for 4 hrs. The culture media containing DOX was then removed by
aspiration, and the cells were washed with control media. Cr transport
assays were performed after growth in control media for 0, 4, 12, 24 or
48 hrs.

[0068] For assays that measured Cr transport kinetics, the final Cr
content in the uptake buffer ranged from 5 to 305 μmol/L. Uptake was
terminated by aspiration of the radiolabeled solution, followed by 3
washes with ice-cold choline buffer. The cells were lysed with 500 mmol/L
NaOH and heated to 80° C. for 30 min. 100 μl of cell lysate was
subjected to scintillation counting using a BECKMAN COULTER LS 6500
scintillation counter. Cr transport was normalized to the protein
concentration measured using the bicinchoninic acid (BCA) protein assay
(PIERCE BIOTECHNOLOGY, Rockford, Ill.).

[0069] For experiments with RNCM, each condition was tested in
quadruplicate. Measurements were performed in triplicate for experiments
performed using HL-1 cells. The number of independent experiments used
for data analysis is noted in each corresponding figure legend.

[0070] DOX Competition Assays:

[0071] For experiments designed to determine if DOX competes with Cr
during the transport cycle, uptake assays were performed in the presence
of 2 μmol/L DOX, or 2 μmol/L DOX and 100 μmol/L B-GPA, a
structural analog of Cr and a inhibitor of Cr transport with an IC50
of 50 μmol/L (Dai W. et al., Arch Biochem Biophys 361: 75-84, 1999).
B-GPA and/or DOX were added to the radiolabeled sodium uptake buffer,
with Cr concentrations ranging from 5-305 μmol/L. Cells were further
processed as described herein above.

[0072] Creatine Transporter Protein (CrT) Antibody Generation:

[0073] Monoclonal rat anti-human creatine transporter protein (CrT)
antibodies were developed using a genetic immunization approach (GENOVAC,
Freiburg, Germany). The antigen for the preparation of the antibodies
consisted of the first 60 amino acids of the human CrT. A number of
positive hybridoma clones were isolated. Two distinct clones (4B9 and
8A6), which only detected the human isoform of CrT, were used for the
studies described herein.

[0077] HL-1 cultures were grown in 75 cm2 plates and transiently
transfected as described for human CrT cDNA. Cell surface proteins were
biotinylated as described by Daniels and Amara (Daniels G. M. & Amara S.
G., Methods Enzymol 296: 307-318, 1998) using the cell surface protein
isolation kit from PIERCE (Rockford, Ill.). Briefly, cell cultures were
washed twice and then resuspended in ice-cold PBS. Cells were lysed with
RIPA buffer (150 mmol/L NaCl; 1% deoxycholate; 1% NP-40; 0.1% SDS; and 10
mmol/L Tris-HCl, pH 7.4) containing protease inhibitors for 25 min.
Samples were centrifuged at 4° C. for 15 min at 15,000 g.
Biotinylated proteins were isolated by incubation of the supernatant with
EZ-LINK IMMOBILIZED NEUTRAVIDIN beads (THERMO SCIENTIFIC, Rockford, Ill.)
for 60 min at room temperature. After extensive washing, the neutravidin
beads were resuspended in Laemmli sample buffer containing 100 mmol/L DTT
and eluted at room temperature for one hour. Intracellular
(non-biotinylated) CrT protein was isolated from the flowthrough of the
NEUTRAVIDIN beads by immunoprecipitation using a specific anti-CrT
antibody. Samples were subjected to SDS-PAGE followed by Western blot
analysis, as described above. Cell surface CrT protein fraction
(biotinylated CrT) was then analyzed by Western blotting using an
anti-CrT antibody as described above.

[0078] Quantification of Cr, and High-Energy Phosphates:

[0079] Tissue culture extracts were prepared using a modification of the
method published by Wiseman et al. (Wiseman R. W. et al., Anal Biochem
204: 383-389, 1992). Cultures were grown in 75 cm2 cm dishes. After
incubation in control or experimental media, the cell monolayer was
washed twice and scrapped in ice cold PBS, and were centrifuged 5 minutes
at 500×g. The cell pellets were flash frozen in liquid nitrogen and
stored at -80° C. until extract preparation. 500 μl of 2N
perchloric acid 5 mM EDTA solution was added directly to the tube
containing the cell pellets. The solution froze on contact with the
pellet and was allowed to thaw to ice/water temperature. The pellet was
homogenized, and the slurry was vortexed vigorously followed by
centrifugation at 4° C., 20000×g, for 5 minutes. The
supernatant was neutralized with an equal volume of 2N KOH, 150 mM TES,
and 3M KCl. The potassium perchlorate precipitate was removed by
centrifugation at 4° C., 20000×g, 2 minutes. The supernatant
was collected and transferred to new tubes and stored at -80° C.
AMP, ADP, ATP, and PCr were measured using a modified approach described
by Goutier et al (Goutier W. et al, J Neurosci Methods 188: 24-31, 2010).
In brief, samples were diluted with mobile phase A (10 mmol/L ammonium
bicarbonate buffer adjusted to pH 9.4 with ammonium hydroxide in 20%
acetonitrile in HPLC grade water), filtered through a 3000 M.W. cut-off
device, and directly injected into a LC-ESI-MS/MS system. Chromatographic
separation was accomplished on a SHIMADZU 20A series HPLC (LC) equipped
with ZIC-pHILIC (5 μm, 150×4.6) column (SEQUANT, AB, Sweden).
Mobile phase B was acetonitrile. Flow rate was 0.8 mL/min. Electrospray
ionization (ESI) tandem-mass (MS/MS) detection was performed on an
APPLIED BIOSYSTEMS/SCIEX API 4000 QTRAP instrument. The following m/z
MS/MS transitions were followed: 346/150.8 (AMP), 426/158.8 (ADP),
506/158.8 (ATP) and 511/158.8 (ATP-15N5 (TRC Toronto, Canada),
was used as internal standard for ADP, ATP, and PCr). Calibration samples
containing each analyte in the range of 0.2-50 μM were run before and
after each batch of study samples. Linear relationship signal
(analyte/internal standard) vs nominal concentration was found for all
the analytes and used for quantification. Cr was measured by a modified
creatinine assay (Il'yasova D et al., Cancer Epidemiol Biomarkers Prev
19: 1506-1510, 2010) on the same equipment as for phosphorylated
metabolites described above. Briefly, samples were diluted with mobile
phase A (10 mmol/L ammonium formate in 0.1% formic acid pH 2.6), filtered
through a 3000 M.W. cut-off device, and 10 μL directly injected into
the LC-ESI-MS/MS system. Chromatographic separation was accomplished on
AGILENT, ZORBAX ECLIPSE PLUS C18, 50×4.6 mm, 1.8 μm column with
the following eluents. Mobile phase B: acetonitrile; flow rate: 1 mL/min.
The following m/z MS/MS transitions were followed: 114/44 (Cr) and 117/47
(Cr--2H3. CAMBRIDGE ISOTOPE LABORATORIES, Maine, USA).
Calibration samples containing Cr in the range of 0.2-500 won were run
before and after each batch of study samples. Linear relationship signal
(analyte/internalstandard) vs nominal concentration was found and used
for quantification. Under the acidic conditions of the assay PCr
undergoes complete hydrolysis to Cr. Thus, measured Cr values were
corrected by subtracting the measure PCr levels under basic conditions.

[0080] Cytotoxicity and Apoptosis Assays:

[0081] RNCM or HL-1 cells transfected with CrT were incubated with 50 or
100 nmol/L DOX for up to 48 hrs. Toxicity was assessed by quantifying the
amount of lactate dehydrogenase (LDH) present in the culture media using
PROMEGA's CYTOTOX96 NON-RADIOACTIVE ASSAY (PROMEGA, Madison, Wis.).
Maximum cytotoxicity was determined by measuring the amount of LDH
released in cells incubated with the kit's lysis buffer. The percentage
of LDH released after DOX treatment was compared with that of controls.

[0082] For apoptosis detection, HL-1 and RNCM cultures were incubated with
100 nmol/L DOX for up to 48 hrs as described above. Apoptosis was
measured using PROMEGA's CASPASE-GLO 3/7 ASSAY SYSTEM (PROMEGA). Briefly,
attached cells in a single 24-well plate were washed 3 times with
ice-cold PBS and resuspended in 500 μl of 1% TRITON-X100; 10%
glycerol; 2 mmol/L EDTA; 137 mmol/L NaCl; and 20 mmol/L Tris, pH 8.0. A
mixture of 25 μL of lysate and 25 μL of caspase 3/7-assay reagent
was prepared, and sequential luminescent readings were taken every 15 min
for 4 hrs. Peak signal readings were normalized to protein content and
plotted.

[0083] Statistical Analyses:

[0084] Data are reported as the mean±standard error of the mean.
Michaelis-Menten plots were generated using curve-fitting software
(SIGMAPLOT, ver. 9.0 San Jose, Calif.). Data were analyzed using
nonlinear, least-squared fitting and ANOVA followed by a Fisher's test
for pair wise, intergroup comparisons (STASTISTICA, ver. 6.0, Tulsa,
Okla.). Correlation was assessed using the Pearson test. Probability
values less than 0.05 were considered significant.

[0085] DOX Reduced Cr Transport in RNCM:

[0086] The effects of DOX on Cr transport capacity were quantified by
measuring 14C--Cr uptake in primary RNCMs. Cr transport decreased
significantly within 2-4 hrs of exposure to 100 nmol/L DOX (FIG. 1A). To
determine if the effect of DOX on Cr transport remained after DOX was
removed from the culture media, Cr transport was measured at increasing
time intervals after an initial exposure of 4 hrs to 100 nmol/L DOX. Cr
transport activity did not recover even after 48 hrs of growth in media
devoid of DOX (FIG. 1B).

[0087] DOX Effects on Cr Transport in HL-1 Cells were Dose and Time
Dependent:

[0089] The dose dependence of DOX's effect on Cr transport was measured in
HL-1 cells following incubation for 24 hrs in media containing 25, 50, 75
or 100 nmol/L DOX. The decrease in Cr transport induced by incubation
with DOX was dose dependent. A significant decrease in Cr transport was
observed in cells incubated for 24 hrs in media containing DOX
concentrations as low as 50 nmol/L (FIG. 2A). The time dependence of
DOX's effects on Cr transport in HL-1 cells expressing the CrT protein
was examined by incubating cultures in 100 nmol/L DOX for 12, 24, 36, and
48 hrs (FIG. 2B). Cr transport decreased by 34.2% (p<0.05) after 24
hrs of exposure and continued to fall through 48 hrs of observation, when
transport was then maximally decreased by 53.5% (p<0.05) relative to
controls.

[0090] Changes in Cr Transport were not Due to Cell Death:

[0091] To determine the contribution of DOX induced cellular injury to the
reduction in Cr transport observed in RNCM or HL-1 cultures, apoptosis
and cytotoxicity were measured in cells incubated with 100 nmol/L
DOX--double the concentration where significant decreases in Cr uptake
were evident (FIG. 2A). Apoptosis was assessed by quantification of
caspase 3/7 activation. No increase in apoptosis was detected in RNCM or
HL-1 cultures incubated with 100 nmol/L DOX for up to 48 hrs (FIG. 3A).

[0092] Cytotoxicity was quantified using LDH release assays. 24 to 36 hrs
elapsed before any changes in LDH release were detected in RNCM cells
incubated in 100 nmol/L of DOX. A small increase in LDH release (7%
compared with control, p<0.05) was detected in RNCM cells after 36 hrs
of exposure to 100 nmol/L DOX (FIG. 3B), 32 hrs after Cr uptake had
decreased by 37% (FIG. 1A).

[0093] In HL-1 cells, 24 hrs incubation with the highest dose of DOX
studied (100 nmol/L) (FIG. 3B) increased LDH release by approximately 10%
compared with controls. This dose of DOX decreased Cr uptake by 34% to
42% (FIGS. 2A and 2B), compared with the 31% reduction measured in these
cells when they were exposed to 50 nmol/L of DOX (FIG. 2A), a dose not
associated with increased cell death in HL-1 nor RNCM cultures (FIG. 3B,
insert).

[0094] DOX Reduced the Vmax and Km for Cr Transport and the
Amount of CrT Protein at the Cell Surface:

[0095] Kinetic analysis of Cr transport measured in HL-1 cells following
12 or 24 hrs of incubation with 100 nmol/L DOX demonstrated a significant
reduction in Vmax. A significant reduction in Km was also
observed after 24 hrs of incubation with DOX (see Table 1 below). To
address the possibility that the reduction in Cr transport was due to DOX
competing for transport with Cr, we determined Vmax and Km
after 14C--Cr uptake assays in the presence of 2 μmol/L DOX or 2
μmol/L DOX and 100 μmol/L B-GPA, a structural analogue of Cr, that
inhibits Cr transport with an IC50 of 50 μmol/L (Dai W. et al.,
Arch Biochem Biophys 361: 75-84, 1999). Acute exposure to DOX did not
significantly alter Cr transport Vmax or Km (see Table 2
below), demonstrating that DOX does not compete with Cr for transport by
CrT.

[0096] Changes in Vmax can indicate changes in the cell surface
population of membrane transporter protein (Qian Y. et al., J Neurosci
17: 45-57, 1997; Zapata A. et al., J Biol Chem 282: 35842-35854, 2007).
Thus, the amount of CrT in the cell membrane was determined using
cell-surface biotinylation followed by avidin affinity binding. This
approach has successfully been used to identify the cell membrane
dwelling population of CrT protein (Darrabie M D et al., Am J Physiol
Endocrinol Metab 300: E870-876, 2011; Li H. et al., Am J Physiol Renal
Physiol 299: F167-177, 2010). CrT protein was detected by Western
blotting using human-specific rat monoclonal antibodies that were
generated using genetic immunization technology as described herein
above. Several distinct antibody clones detected the N-terminus of the
human isoform of the CrT, with a 55 kD band corresponding to the monomer
and cell-surface CrT protein. The heavier CrT bands likely represent
adducts (multimers) of the transporter protein. No CrT specific bands
were detected in cells transfected with a negative control, or in the
cells that expressed a chimeric rat/human CrT protein where the
N-terminus was identical to that of the rat isoform (FIG. 4).

[0097] The amount of CrT at the cell surface was decreased in HL-1 cell
cultures grown in media with 100 nmol/L DOX (FIGS. 5A and 5B). These
changes in CrT protein mirrored the decrease in Cr transport capacity
observed after exposure to DOX; there was a positive correlation (r=0.69,
p<0.05, n=12) between Vmax (Table 2) and cell-surface CrT protein
abundance.

[0099] The effects of DOX on intracellular content of Cr, PCr, ATP, ADP
and AMP were quantified by LCMS/MS analysis performed on extracts
prepared from HL-1 cultures incubated in the presence of 100 nmol/L DOX
for 24 hrs and compared with data obtained from control cultures. The
only significant difference measured was a decrease in AMP in cultures
exposed to DOX (see Table 3 below).

[0100] Formalin-fixed tissue samples of cardiac tissue obtained from (i)
transgenic mice overexpressing the human creatine transporter protein
(CrT) (FIG. 6); (ii) human subjects (FIG. 7); or (iii) human subjects
having either normal cardiac tissue or failing cardiac tissue (tissues
were obtained under an approved IRB protocol) (FIG. 8) were embedded in
paraffin. Four micrometer sections were cut from the tissue blocks,
placed on positive charged slides, allowed to dry, and then heated in a
65° C. oven for thirty minutes. After removal of paraffin with
xylene and clearing with alcohol, the slides were placed in hydrogen
peroxide and methanol to quench endogenous peroxidase activity. Sections
were hydrated and washed in deionized water. It was determined during
antibody optimization that pretreatment with proteinase K (DAKO
concentrate diluted to 0.05 ml in 1.0 ml of 0.05M Tris, pH 7.5) was the
proteolytic enzyme of choice. Comparative tissue pretreatment studies
were performed using heat induced epitope retrieval. The tissue sections
were digested for five minutes in the proteinase K solution then rinsed
in deionized water and placed in Tris buffered saline (TBS) pH 7.5.
Primary antibody rat monoclonal anti-4B9 or anti-8A6 at dilutions of 1:25
to 1:500 were applied and incubated for one hour at room temperature (the
4B9 and 8A6 antibodies were generated as described herein above at
Example 1 in the section Creatine Transporter Protein (CrT) Antibody
Generation). Following a rinse and wash with TBS, the bound primary
antibody was linked with biotinylated goat anti-rat IgG (H&L specific, 5
μg/ml, VECTOR LABORATORIES, Burlingame, Calif.). The formed immune
complex was further amplified and labeled with horseradish peroxidase
conjugated avidin biotin complex (ABC ELITE, VECTOR LABS).
Diaminobenzidine (DAB) was used to visualize the complex consisting of
the bound anti CrT antibodies-biotin-avidin horseradish peroxidase The
slides were washed with tap water, hematoxylin counter stain was applied,
followed by dehydration with absolute alcohol, cleared with xylene and
cover slipped with a permanent mounting media.

Example 3

Measurement of Creatine Transport in Erythrocytes

[0101] A sample of human whole blood was collected and creatine transport
was quantified according to the following procedure. First, whole blood
(10 ml) was centrifuged at 1500 g for 5 minutes and white nucleated
elements were removed by aspiration of the buffy coat (Berthiaume J. M. &
Wallace K. B., Cell Biol Toxicol 23: 15-25, 2007). Cells were resuspended
in RPMI (INVITROGEN, Carlsbad, Calif.) media and plated in 24 well
culture plates. To measure creatine transport, cells were centrifuged in
the culture dishes at 1500 g, the supernatant was removed by aspiration,
and the cells were resuspended in room temperature choline buffer (150
mmol/L choline chloride; 1 mmol/L CaCl2; 5 mmol/L MgCl2; 2
mmol/L KCl; and 5 mmol/L HEPES-Tris, pH 7.5). This procedure was repeated
once more. After the last centrifugation, the cells were resuspended in
sodium uptake buffer (150 mmol/L NaCl; 1 mmol/L CaCl2; 5 mmol/L
MgCl2; 2 mmol/L KCl; and 5 mmol/L HEPES-Tris, pH 7.5) that was
supplemented with 0.275 μCi/ml of 14C--Cr (55 mCi/mmol, AMERICAN
RADIOLABELED CHEMICALS, St Louis, Mo.) and unlabeled Cr to a final
concentration of 15 μmol/L for the dose-response and time course
experiments, which is within the reported physiological range (Persky A.
M., et al., J Clin Pharmacol 43: 29-37, 2003). Cells were then incubated
for 10 min in a CO2 incubator at 37° C. Uptake was terminated
by removal of the radiolabeled solution by centrifugation as described
above and three washes in ice cold choline buffer also as described
above. Since creatine transport depends on the physiological sodium
gradient, non-specific creatine uptake was measured in cells that were
incubated in choline buffer supplemented with radiolabeled creatine. Data
obtained following these experimental conditions represents non-specific
creatine binding/absorption that does not involve the CrT. The cells were
lysed with 500 mmol/L NaOH and heated to 80° C. for 30 min. 100
μl of cell lysate was subjected to scintillation counting using a
BECKMAN COULTER LS 6500 scintillation counter. Cr transport was
normalized to cell number, determined by manual counting using a
hematocytometer. The results are shown in FIG. 9.

[0102] Creatine transporter protein present on the surface of erythrocytes
from human whole blood samples was detected using a monoclonal antibody
specific for creatine transporter protein. First, the cells were
collected and processed prior to antibody detection according to the
following procedure. Whole blood was centrifuged at 1500 g for 5 minutes
and white nucleated elements were removed by aspiration of the buffy
coat. Isolated erythrocytes were smeared on glass slides and let dry at
room temperature, then fixed with 100% methanol for 30 minutes.
Erythrocytes were probed overnight with 1:100 4B9 or 8A6 (the antibodies
were generated as described herein above at Example 1 in the section
Creatine Transporter Protein (CrT) Antibody Generation). The slides were
washed three times with PBS, before being probed for 1 hour with a 1:500
dilution of ALEXA FLUOR-GOAT anti-rat IgG secondary antibody, and washed
again. Erythrocytes probed with only the secondary antibody were used as
a negative control.

[0103] Slides were visualized and photographed using a fluorescence
microscope, with the blue 470 nm or 490 nm filter (see FIG. 10).
Erythrocytes shown in FIG. 10 were immunolabeled using 8A6 antibody,
whereas those shown in FIG. 11 were labeled using 4B9 antibody. The image
in FIG. 11 is magnified to depict in greater detail the fluorescent
signal on the cell surface of the erythrocytes.

[0104] Variations and modifications of the herein described systems,
apparatuses, methods and other applications will undoubtedly suggest
themselves to those skilled in the art. Accordingly, the foregoing
description should be taken as illustrative and not in a limiting sense.

[0105] Any patents or publications mentioned in this specification are
indicative of the levels of those skilled in the art to which the
invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.